Herpes simplex virus 1 open reading frames O and P are not necessary for establishment of latent infection in mice

Open reading frame (ORF) O and ORF P partially overlap and are located anti sense to the gamma(1)34.5 gene within the domain transcribed during latency . In wild-type virus-infected cells, ORF O and ORF P are completely repress ed during productive infection by ICP4, the major viral transcriptional act ivator/repressor. In cells infected with a mutant in which ORF P was derepr essed there was a significant delay in the appearance of the viral cu-regul atory proteins ICP0 and ICP22. The ORF O protein binds to and inhibits ICP4 binding to its cognate DNA site in vitro. These characteristics suggested a role for ORF O and ORF P in the establishment of latency. To test this hy pothesis, two recombinant viruses were constructed. In the first, R7538(P-/ O-), the ORF P initiator methionine codon, which also serves as the initiat or methionine codon for ORF O, was replaced and a diagnostic restriction en donuclease was introduced upstream. In the second, R7543(P-/O-)R, the mutat ions were repaired to restore the wild-type virus sequences. We report the following. (i) The R7538(P-/O-) mutant failed to express ORF O and ORF P pr oteins but expressed a wild-type gamma(1)34.5 protein. (ii) R7538(P-/O-) yi elds were similar to that of the wild type following infection of cell cult ure or following infection of mice by intracerebral or ocular routes. (iii) R7538(P-/O-) virus reactivated from latency following explanation and cocu ltivation of murine trigeminal ganglia with Vero cells at a frequency simil ar to that of the wild type, herpes simplex virus 1(F). (iv) The amount of latent R7538(P-/O-) virus as assayed by quantitative PCR is eightfold less than that of the repair virus. The repaired virus could not be differentiat ed from the nild-type parent in any of the assays done in this study. We co nclude that ORF O and ORF P are not essential for the establishment of late ncy in mice but may play a role in determining the quantity of latent virus maintained in sensory neurons.